Chronic Myeloid Leukemia (CML) is a disorder of hematopoietic cells wherein the appearance of the Philadelphia chromosome (a translocation of chromosomes 9 and 22) results in expression of the fusion oncoprotein BCR-ABL, whose disregulated tyrosine kinase activity causes the abnormal myeloid cell growth characteristic of the disease. In the last 20 years, substantial advances have been made in targeting BCR-ABL therapeutically to treat CML; current therapy for the disease involves daily administration of the tyrosine kinase inhibitor Imatinib Mesylate (Gleevec) and/or several second-generation inhibitors (Dasatinib, Nilotinib). However, increasing resistance to the current line of ATP-competitive inhibitors of BCR-ABL warrants discovery of novel therapeutics that can target all BCR-ABL proteins with high specificity. Recently the potential of using fusion junction-targeted small interfering RNA (siRNA) to silence BCR-ABL has been demonstrated in vitro and ex vivo. This technology could be used to design a powerful line of therapeutics that suppresses all variants of activated Abl, even imatinib/dasatinib resistant forms. But advancement of this approach toward gene silencing in vivo requires developing methods for optimizing junction-targeted siRNA efficiency, specificity, and delivery. Aim 1 of this proposed research is to design a potent and specific junction-targeted small silencing RNA for suppression of the predominant transcript variant of BCR-ABL (b3a2) and to apply techniques to load this construct into polymeric nanoparticles for delivery. In Aim 2, strategies for enhancing delivery of polymeric nanoparticles to leukemic cells via surface modification will be explored using a micro-scale fluid flow device that mimics nanoparticle-cell interactions expected in the circulatory system. After optimization of the small silencing RNA and its delivery vehicle, Aim 3 seeks to assess the efficacy of the engineered therapeutic in a humanized mouse model of leukemia. Through these aims, significant progress will be made toward the discovery and optimization of an RNAi based therapeutic for more broad, but equally potent, treatment of CML.